Pharmaceutical compositions for use in the treatment of wounds

ABSTRACT

A pharmaceutical composition that can be used in the treatment of wounds, comprising at least a sulfated polysaccharide and wherein the polysaccharide/s are in the form of solids as powder, flakes or sheets obtained through a lyophylization process. Particularly, this invention relates to the use of one or more sulfated polysaccharides, as solids in the form of sheets obtained by lyophylization, in the healing of wounds in humans and animals.

The present invention relates to a pharmaceutical composition that canbe used in the treatment of wounds, that comprises at least one sulfatedpolysaccharide and wherein the polysaccharide/s are in the form of asolid as powder, flakes or sheets obtained by a lyophylization process.Specifically, the invention relates to the use of one or more sulfatedpolysaccharides, as a solid in the form of sheets obtained bylyophylization, in the healing of wounds of humans and animals.

BACKGROUND OF THE INVENTION A) Current Treatments to Stimulate theGranulation Tissue

Different kinds of treatments have been proposed to stimulate thegranulation tissue, which is essential to close a wound, as this highlyvascularized tissue fills the defects and prepare the surface forepithelization. Among the proposed approaches, we can mention:

a) Treatments to stimulate the granulation tissue: different approachesare being assayed to stimulate the healing processes, being among themost promising ones those using mediators, for example, the recombinanthuman growth hormone. (“Effects of recombinant human growth hormone ondonor site healing in severely burned children”, Herndon D. N. et al.;1990; 212: 424-431 and “Recombinant human growth hormone accelerateswound healing in children with large cutaneous burns”, Gilpin D. A. etal.)

b) Treatments to diminish and module the excessive development ofgranulation tissue: in the literature it is described the use oftopications with 1% silver nitrate. Said compound formulated in the formof a bar is applied in solid state, rubbing the surface of the abundantgranulation tissue that hinders the later ephitelization process. Inthis way, leveling of granulations is sought by the cauterization of thesame, thus allowing the advance of the epithelial front (“Historicalreview of the use of silver in the treatment of burns”, Klosen H. J. andBurns, March 2000 and “Reconstructive surgery of the injured nail”,Swanker W. A; The American Journal of Surgery, September 1947).c) Local use of corticoids: corticoids act as atrophogenic substances,blocking the inflammatory processes involved in the tissue reparation.Therefore, they decrease the presence and advance of “granulations” orangiogenesis. (“Topical potent corticosteroids for excessive granulationtissue”, Nordir, P. C. et al., Dermatol. Surg. Dec. 24, 1998(12):1409-10 and “Topical diflorosone ointment for treatment ofrecalcitrant, excessive granulation tissue”, Mandrea, E; Dermatol. Surg.Jun. 25, 1999 (6):517)d) Surgical procedures: they comprise scraping off the hypertrophicgranulation tissue by scalpel. Due to the trauma caused by this method,the same must be executed in an operating room under anesthesia. (“Fromwound to scar”, Linares, H. A.; Burns, Vol.22, no5, pp. 339-352, 1966)

B) Evaluation of the Current Treatments to Stimulate the GranulationTissue

However, none of the above-mentioned methods is sufficiently efficient.This is due mainly to the following reasons:

-   -   a) The silver nitrate produces a chemical destruction of the        hypertrophic granulation leaving a contaminated bed, with        residues of the product and a secreting trend. In addition, its        application frequently is painful and generally must be repeated        several times. This compound has no direct action on the        vitality of the granulation tissue.    -   b) Corticoids, due to their atrophogenic action, not only they        would tend to diminish the abundance of the tissue under        repairing, but also interfere with the process of forming the        same, decreasing its vitality and delaying the cellular        migration. In the practice, a relative effect is observed with a        decrease of the “hypertrophic granules” (insufficient action).        In addition, if topication is intense, interference in        reparation may be observed.    -   c) The surgical procedure of scrapping off the granulations        using a scalpel, besides being traumatic and troublesome (the        patient must be under deep anesthesia or anesthesia) is executed        as a previous step to a skin replacement (auto-graft)        C) Products Proposed to Stimulate the Granulation Tissue.        Sulfated Polysaccharides

The polysaccharides are polymeric substances formed by units ofmonosaccharides. There is a great variety of linear or branchedpolysaccharides, which may be formed by only one type of monosaccharidesor different types of monosaccharides. Starch and cellulose are veryimportant members of this kind of compounds. [Aspinall G. O., (1982)“General Introduction” in “The Polysaccharides” (Aspinall G. O.,Ed.)Academic Press, Vol I, page 1-17].

Either in the vegetal kingdom, as in the animal kingdom, there existpolysaccharides wherein some hydroxyls are substituted forming sulfateesters. Examples of this kind of polysaccharides are glycosaminoglycanssuch as heparin, heparan sulfate, etc. which are present in all animaltissues [Fransson L. A., (1988) “Mammalian Polysaccharides” in “ThePolysaccharides” (Aspinall G. O., ed.) Academic Press, Vol III,p.338-406). Also the structure of sulfated polysaccharides has beendetermined (sulfated L-galactans and L-fucans) in marine invertebratesand, recently, in marine angiosperms [M. S. Pereira, F. R. Melo, and P.A. S. Mourão, “Is there a correlation between structure andanticoagulant action of sulfated galactans and sulfated fucans”,Glycobiology, 12, 573-580 (2002); R. S. Aquino, A. M.Landeira-Fernandez, A. P. Valente, L. R. Andrade, and P. A. S. Mourao,“Occurrence of sulfated galactans in marine angiosperms: evolutionaryimplications”, Glycobiology, 15, 11-20 (2005)].

In the algae, they are the principal constituents of the intercellularmatrix and the cellular wall, and in some cases, such as in red algae,they represent more than 50% dry weight of the alga. [Painter T. J.,(1988) “Algal polysaccharides” in “The Polysaccharides” (Aspinall G. O.,ed.) Academic Press, Vol II, page 196-27].

It has been demonstrated that these polysaccharides have a great varietyof biological properties, such as antiviral, antitumor, antioxidant,anticoagulant, antithrombotic activities, etc. [Damonte E. B.,Matulewicz, Cerezo A. S., (2004) “Sulfated seaweed polysaccharides asantiviral Agents”, Current Medicinal Chemistry, 11, 2399-2419].[AlbanS., (1997) “Carbohydrates with anticoagulant and antithromboticproperties” in “Carbohydrates in Drug Design” (Witczak Z. J. andNieforth K. A., eds.) Marcel Dekker, Inc. page 209-277] [Witvrouw M.,Pannecouque C., De Clercq E., (1997) “Polysulfates: Chemistry andPotential as Antiviral Drugs” in “Carbohydrates in Drug Design”; I. M.Yermak and Y. S. Khotimchenko, in Recent Advances in MarineBiotechnology, Vol. 9 Biomaterials and Bioprocessing, ed. M. Fingermanand R. Nagabhushanam Science Publishers Inc. 2003, Enfield (N.H.) USA,“Chemical Properties, Biological activities and applications ofcarrageenans from red algae”, p. 207-255]

However, not all of these compounds show the same degree of activity andtheir behavior is related to the position and degree of sulfatation. Inaddition, it has been demonstrated that artificially sulfatedpolysaccharides and even sulfated synthetic polymers show, in somecases, similar activities. [Damonte E. B., Matulewicz, Cerezo A. S.,(2004) “Sulfated seaweed polysaccharides as antiviral Agents”, CurrentMedicinal Chemistry, 11, 2399-2419] [Alban S., (1997) “Carbohydrateswith anticoagulant and antithrombotic properties” in “Carbohydrates inDrug Design” (Witczak Z. J. and Nieforth K. A., eds.) Marcel Dekker,Inc. page 209-277.] [Witvrouw M., Pannecouque C., De Clercq E., (1997)“Polysulfates: Chemistry and Potential as Antiviral Drugs” in“Carbohydrates in Drug Design”; I. M.].

The sulfated galactans comprise the cellular matrix of most of the redalgae. They are linear polysaccharides formed by alternating units ofβ-D-galactopyranose bound by position 3 andα-(3,6-anhidro)galactopyranose bound by position 4. Polysaccharideswherein units α are in configuration D are known as carrageenans, whilewhen this unit is in configuration L, they are aragans, being agarosethe most known member of this family of polysaccharides, that is notsulfated. The agarans and carrageenans are sets of differentpolysaccharides having similar hydrocarbon skeletons and different typeand position of substitution, which results in important variations intheir physical properties and biological activity. Thus, carrageenansare classified in different families according to the function of thesulfatation position in the β-galactose unit. Carrageenans of the kappafamily are those wherein this unit is sulfated in C-4, kappa and iotacarrageenans belong to this family. Moreover, lambda carrageenans arecharacterized by the sulfatation in C-2 of the unit β, being the mostimportant member of this family the lambda carrageenan (FIG. 1). Besidesthe sulfatation position, the kappa carrageenan has only one sulfategroup by repetitive disaccharide unit, the iota two and the lambda,three. Kappa and iota repeating disaccharide units may coexist in thesame carrageenan, which is indicated as a kappa/iota hybrid. [C. A.Stortz and A. S. Cerezo, “Novel findings in carrageenans, agaroids and“hybrid” red seaweed galactans”, Current Topics in Phytochemistry, 4,121-134 (2000)].

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a pharmaceutical composition that canbe used in the treatment of wounds, comprising at least one sulfatedpolysaccharide and wherein the polysaccharide/s is/are as solids in theform of powder, flakes or sheets. Specifically, the invention relates tothe use of sulfated polysaccharides, as solids in the form oflyophilized, dehydrated and sterilized sheets, over traumatic cutaneouswounds, such as, for example, intermediate and deep burns, and overnon-traumatic wounds, such as venous ulcers.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be described in connection with the attacheddrawings, in which:

FIGS. 1A-1D illustrate the most important structures of red algagalactans;

FIGS. 2A-2C illustrate Lyophilized carrageenans prepared according tothe described procedure; and

FIGS. 3-12 are photographic images of right lower third of an arm, elbowand upper third of a forearm, of a patient illustrating treatment of awound over time.

DETAILED DESCRIPTION OF THE INVENTION

In a particular embodiment of the invention, from the group of sulfatedpolysaccharides, preferred members are kappa or iota carrageenans orhybrids thereof and lambda carrageenans obtained from red algae of thespecies Iridaea, Gigartina, Eucheuma and Kappaphyccus.

Closing of wounds occurs when, among other multiple and complexbiological processes, the granulation tissue advances over the bed ofwounds, as a protagonist of the intermediate step of healing. Thegranulation tissue is a highly cellular, vascular and functionalstructure primarily devoted to clean the damaged area, fills theexisting tissue defect and supports the development of new and activetissue of the mesenchyme. However, the bed of a wound may show differentaspects that reveal the status and the course of action of this tissueand scarring development. In relation with the granulation tissue, ananatomoclinic distinction between at least two forms of presentation ofthe bed under repairing has to be made:

-   1) A wound with a flat, pale and low vital bed, that tends to delay    appearance of the suitable granulation tissue to start the process    of repairing by production of the extracellular matrix; such as, for    example: the bed of a deep intermediate burn (classification from    the American Burn Association) (or type AB according to the    classification of Benaim); or torpid scar beds as in venous ulcers    and similar.-   2) A wound with hypertrophic granulation tissue (uncontrolled    growth). The excess of granulation tissue (hypertrophic granulation)    prevents the potential epithelization action (progression of    epithelial cells from anexial reservoirs and/or the cutaneous edges    of a wound) spreads covering a bleeding bed with epithelial cells    devoted to close the wound.

The inventors of the present invention have found that theabove-mentioned cases can be successfully treated applying thecomposition of the invention.

The present invention relates to a pharmaceutical composition that canbe used in the treatment of wounds, comprising at least a sulfatedpolysaccharide and wherein the polysaccharide/s is/are in a solid formas powder, flakes or sheets obtained by a lyophylization, dehydrationand sterilization process. Particularly, the invention relates to theuse of solid sulfated polysaccharides, such as sheets obtained bylyophylization, dehydration and sterilization, over traumatic cutaneouswounds, such as for example intermediate and deep burns, andnon-traumatic wounds, for example venous ulcers.

In a particular embodiment of the invention, from the group of saturatedpolysaccharides, kappa or iota carrageenans or hybrids thereof arepreferred, as well as the lambda carrageenans obtained by extractionwith water at ambient temperature or heat and removing the products oflow molecular weight, of red algae of the species Irideae, Gigartina,Eucheuma and Kappaphyccus.

Carrageenans are compounds that are industrially extracted with alkalineaqueous solutions of different species of red algae (rodoficeae) usuallypretreated also with alkali, and used in different industries such asthe food and cosmetic industries, etc. as thickening and/or gellingagents. Carrageenans obtained under these conditions require heating todissolve in water, but when cooled form gels, which rheologicalparameters depend on the concentration of polymer and number ofpotassium, ammonium or calcium ions contained by the hydrocolloid.

Thus, a wound with a flat, pale and low vital bed, that tends to delayappearance of the suitable granulation tissue to start the process ofrepairing by production of the extracellular matrix, could be treatedwith the composition of the invention, by applying locally sheets oflyophilized, dehydrated and sterilized carrageenans over the devitalizedbed of the wounds by the scrapping procedure.

Moreover, a wound with hypertrophic granulation tissue, could be treatedwith the composition of the invention, by applying topically the samepreparations over the hypertrophic granulation tissue, allowing in thisway to control its volume and harmonize development of the same.

Both application procedures are to increment vitality and control thedevelopment of the repairing tissue producing in a lesser time theextracellular matrix and closing of the wound. The contact ofcarrageenans, prepared as previously described, with the surface totreat produces a sensible adherence to the same, and so the scrappingoff procedure must be executed firmly, obtaining a bleeding of the bedpartially due to the traumatic action over the cellular coating ofgranulation tissue on which the carrageenan preparation has “adhered”,and partially to an anticoagulant property of the carrageenan. Thebeneficial effects would be the result of the contact of blood with allthe surface of the wound, transporting growth and stimulating factors;as well as of an own and direct action of the carrageenan preparation,as the simple scrapping mechanism does not attain the effects hereinobtained.

This form of use usually is complemented with a later application ofsaid preparation in the form or sheets, that are left as a biologicaldressing over the treated surfaces between 24 to 72 hours, and as acomplement of the previous treatment. In this way, it is possible toreduce progressively the abundance of an exceeded granulation tissue,allowing the advance of the epithelium from the extensions and the edgesof the wound treated to complete healing.

EXAMPLE

The carrageenan samples used in the first case correspond to akappa/iota hybrid carrageenan extracted from cystocarpic specimens ofthe red alga Gigartina skottsbergii, collected in Bahia Camarones,Chubut (Argentina).

The agarose, which is a neutral polysaccharide, without sulfate groups,but with a chain similar to the carrageenans (the unit of α-galactose isin L configuration instead of D) showed no activity and the results withsodium alginate were not conclusive. Notwithstanding the latterpolysaccharide is acid, it does not have sulfate groups and its acidityis due to the presence of the carboxyl groups of the uronic acidsforming the same.

-   1) Procedure to prepare sheets of lyophilized, dehydrated and    sterilized carrageenans from cystocarpic specimens of red alga    Gigartina skottsbergii:

The dry alga was grinded using a Wiley mill (20-mesh screen).Thereafter, the grinded alga (50 g) was suspended in water (2,5 1) andextracted under mechanical stirring at ambient temperature during 16hours. The mixture was centrifuged at 8000 rpm at a temperature of 10°C. and the supernatant was concentrated to half of its volume removingthe products of low molecular weight by dialysis (MWCO 12-14000). Thesolution was lyophilized in plastic trays of suitable dimensions andforms, obtaining a spongy product of high contact surface, of about 1-2cm of height and a surface of 10×10 cm, which was later dehydrated andsterilized, and fractioned as needed (FIG. 2).

It is very important that the solution to lyophilize has such a volumeand concentration that the final lyophilized, dehydrated and sterilizedproduct has the largest contact surface and the adequate consistency soas to be applied to the wound, that is scrapped off as previouslydescribed, and then the product is left, as a biological dressing, onthe wound during 72 hours without disintegrating.

-   2) Treatment of wounds using carrageenans    -   Devitalized or hyper granulating beds (photograph number 1:        image of right lower third of an arm, elbow and upper third of a        forearm, of a patient of 50 years old burnt with hot water 16        days ago, resulting in a lesion equivalent to 2% of the body        surface, type deep intermediate and deep according to the        classification of the American Burn Association [or AB and B        according to the classification of Benaim]. At [“A”] rests of B        or deep scar with devitalized and necrobiotic tissue are        observed, and at “B” incipient pale and hypertrophic granulation        tissue).    -   Application of carrageenans in powder, flake or sheet forms        (photograph number 9 shows the use of pieces of carrageenans        over the burn of another patient), by scrapping (photograph        number 10 with the same patient showing the surface already        stimulated and bleeding resulting from the above described use).    -   Stimulation and control of granulation tissue development        (photograph number 2 of the patient initially selected at day 7        and after receiving two applications of carrageenans. It is        observed in [“A”] a complete change in the evolution of a bed        that in spite of showing more reduced rests of B scars, it is        controlled by the development of a suitable granulation tissue        allowing the advance of marginal [“C”] and eccentric [“B”]        epithelization from the annexial epithelial rests).    -   Improvement of beds with the appearance of convergent epithelial        islands, resulting from stimulating the epithelial reservoir of        non-destroyed extensions (photograph number 3 18 days after the        start of the treatment with an epithelized surface of about        80%). Carrageenans were applied between 48 and 72 hours.    -   Gradual epithelial healing with advance of convergent and        divergent epithelium (photograph number 4 with 90% of the        surface epithelized; though persistent rests of deep beds or B        in late spontaneous removal stage [“A”] are still observed).        Twenty-two days of progress. Photograph number 5 shows the same        tendency, days after ([“A”] shows small rests of scar that have        not been self-removed yet).    -   Complete closing of the wound with reduction of healing times        (photograph number 6), after almost one month of treatment.    -   Reduction of effects (photograph number 7 shows a post-healing        skin having a good consistency and flexibility).    -   Optimization of esthetic and functional healing results        (photograph number 8 taken two months after the start of        treatment).

Conclusions

The conclusions obtained from this example can be extended to othersulfated polysaccharides, as many of them have similar biologicalactivities. Said biological activities are attributed to the presence ofsulfate groups, though they many express to a greater or lesser extentdepending on: a) the degree of sulfatation, b) the position ofsulfatation, c) the linear or branched structure of the compound, d) themolecular weight.

Therefore, it is considered that every sulfated polysaccharide that maybe obtained under the above-described conditions could be used in theclaimed pharmaceutical composition to promote healing of wounds by thedisclosed procedure. The above considerations can be extended to othersulfated polymers that meet the indicated conditions, even though theyare not polysaccharides.

1. A pharmaceutical composition that can be used in the treatment of wounds, characterized by comprising at least a sulfated polysaccharide and wherein the polysaccharide/s are in the form of solids as powder, flakes or sheets obtained lyophilizing, dehydrating and sterilizing solutions of the same.
 2. A pharmaceutical composition that can be used in the treatment of wounds, according to claim 1, characterized by comprising a sulfated polysaccharide and wherein the polysaccharide is in the form of sheets obtained lyophilizing, dehydrating and sterilizing solutions of the same.
 3. A pharmaceutical composition that can be used in the treatment of wounds, according to claim 1, characterized in that the sulfated polysaccharide is selected from kappa, iota, kappa/iota or lambda carrageenans.
 4. A pharmaceutical composition that can be used in the treatment of wounds, according to claim 1, characterized by comprising a mixture of sulfated polysaccharides and wherein at least one of them is in the form of a lyophilized solid.
 5. A pharmaceutical composition that can be used in the treatment of wounds, according to claim 1, characterized in that at least one of the sulfated polysaccharides is a carrageenan.
 6. A pharmaceutical composition that can be used in the treatment of wounds, according to claim 1, characterized in that at least one sulfated polysaccharide in the form of a lyophilized, dehydrated and sterilized powder is used in the preparation of a composition for treating wounds.
 7. A pharmaceutical composition that can be used in the treatment of wounds, according to claim 1, characterized by containing at least one carrageenan in the form of a lyophilized, dehydrated and sterilized solid.
 8. A pharmaceutical composition that can be used in the treatment of wounds, according to claim 1, characterized by containing, at least, a natural or synthetic sulfated polymer in the form of a lyophilized, dehydrated and sterilized solid.
 9. The use of at least one sulfated polysaccharide in the form of a lyophilized solid in the preparation of a composition for treating wounds in humans and animals.
 10. The use of at least one carrageenan in the form of a lyophilized solid in the preparation of a composition for treating wounds in humans and animals. 